Human cytidine deaminases APOBEC3 (A3) proteins are potent host defenses against HIV. These antiviral proteins induce lethal modification of cytosines to uracils in newly synthesized minus-strand viral DNA, resulting in abortive viral infection. HIV must overcome these host cellular defenses for successful viral replication. HIV-1 encodes a protein, Vif, which suppresses the antiviral effects of A3 proteins by targeting them for degradation through the 26S proteasome. Vif hijacks cellular Cullin5 (Cul5), ElonginB, and ElonginC to form a viral E3 ubiquitin ligase that targets A3G for polyubiquitination and degradation. Thus, identification of novel strategies to preserve the antiviral functions of A3 is an exciting new target for antiretroviral therapy. In this application, we propose to capitalize our expertise in HIV-1 Vif/A3 system and our new understanding of the viral evasion mechanism to develop a rapid cell-based assay for the identification of small molecule inhibitors of Vif and to further optimize and adapt the system for application to high throughput molecular screening of large compound libraries to identify molecules that inhibit HIV-1 replication. We will also characterize several small molecule compounds that have been identified to inhibit Vif mediated A3G degradation in our initial screening. PUBLIC HEALTH RELEVANCE: The HIV-1 virion infectivity factor (Vif) is an essential regulatory protein required for HIV- 1 replication in natural target cells such as CD4+ T-cells and macrophages which express innate antiviral human APOBEC3G (A3G) and related cytidine deaminases. We have identified a small molecule inhibitor of Vif. The overall goal of this project is to develop a rapid cell-based assay for the identification of small molecule inhibitors of Vif and to further optimize and adapt the system for application to high throughput molecular screening of large compound libraries to identify molecules that inhibit HIV-1 replication.